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Breeding Polyploid Acacia mangium for wood production

Breeding Polyploid Acacia mangium for wood production. Jane L. Harbard School of Plant Science, University of Tasmania. Tetraploid A. mangium in RCFTI Trial, Vietnam . What is polyploidy?. having three, four, or more sets of chromosomes A. mangium 2n=2x=26

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Breeding Polyploid Acacia mangium for wood production

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  1. Breeding Polyploid Acacia mangium for wood production Jane L. Harbard School of Plant Science, University of Tasmania Tetraploid A. mangium in RCFTI Trial, Vietnam

  2. What is polyploidy? • having three, four, or more sets of chromosomes • A. mangium 2n=2x=26 • 40-70% of plant taxa are polyploid • including many tree species Populus tremula, Fraxinusamericana,Ulmusamericana, Quercusrobur, Betulapubescens, Leucaenaleucocephala, Sequoia sempervirens Casuarinalittoralis 2x, 4x A. cowleana 4x A. holosericea 2x, 4x, 6x A. aneura 3x, 5x

  3. A complicated business - why should we bother? • Australian Acacia are important plantation species in SE Asia including Vietnam • adaptable across sites-growing well on poorer soils, nitrogen fixers • short rotation crop producing pulp wood – 6 years and saw logs at 8-9 years Plantation Area Vietnam (ha) A. auriculiformis: 92,000 A. mangium: 154,000 A.m x A.a hybrid: 232,000 (Ministry of Agriculture and Rural Dev. 2009) • but they are prolific seed producers – can be invasive

  4. Established polyploid breeding programs • Polyploid breeding is a staple of diverse agricultural species such as wheat, sugar cane, hops, cassava, watermelon, citrus • Traits of interest include • seedlessness via seedless triploids • increase in chemical compounds of interest • increased biomass • larger fruits

  5. Tetraploid (4x) and triploid (3x) traits of interest • Gigantism characteristics • longer wood fibres (good for papermaking) • adaptations to wider range of environments ? • Pure species and hybrid triploids - 4x X 2x = 3x • faster growth than 2x (?) • demonstrated in commercial triploid hybrid leucaena for fodder production • sterile …less weedy • biomass reallocated from reproductive to vegetative growth (increased wood production?)

  6. Outline of the program – an Australia/Vietnam collaboration 2x Aa 4x Am 2x Am 32 lines captured, bulked up in TC and weaned for mother hedge plants Vietnam Polyploid lines to RCFTI Vietnam from Shell Forestry LTD. UK 2001 Bau Bang hybridising orchard in south of Vietnam- planted 2003 Test OP progeny from 4x parents Clone and breed from best trees

  7. Generating new 4x genotypes using a range of colchicine concentrations and exposure times • Application of colchicine to apical meristem of germinated seedlings • Seed immersion in aqueous colchicine solution • Seed germination on colchicine saturated filter paper • Treatments applied to bulk seedlots of A. mangium seed supplied by RCFTI seed orchards in Vietnam

  8. Application of colchicine to apical meristem of germinated seedlings “Drop method” 5µl of colchicine applied to apical meristem at seedling emergence at midday for 3 days labour intensive, not all seed treated 8.9% 4x with 1.5% colchicine

  9. Seed immersion in aqueous colchicine solution seed immersion in colchicine solution impaired shoot and root growth not successful for A. mangium Germinants following immersion in 0.01% colchicine solution for 16 h, 27 days after transfer to Petri dishes for germination

  10. Seed germination on colchicine saturated filter paper 0.02% colchicine soaked filter paper, 16 hours at 29 C 7% 4x Seeds following treatment with 0.02% colchicine on saturated filter paper for 16 h and transfer to fresh Petri dish for germination. Germinants 4 days after transfer showing normal root growth

  11. Flow Cytometry • must sample min.2 phyllodes/plant • phyllode sample + modified Galbraith Buffer • Pisumsativumcv. Torstag – internal standard • filtered through a BD Falcon 40 µm nylon cell strainer • propidium iodide stain (20µg/ml)  • Coulter Elite ESP Cell Sorter • known 2x and 4x included each run • 5000 or10000 counts / sample

  12. FCM - output mixoploid 4x 2x 2C DNA amounts – P. sativum (9.10 pg) 2x A. mangium (1.3pg) 4x A. mangium (2.6pg)

  13. Induction 2010-2011 • 250 seed x 5 families from Elite Vietnam genotypes • 0.02% colchicine imbibed on filter paper 29C for ↑24 hours Plants aged 8 months

  14. Ploidy types following colchicine induction 4x mixoploid 2x control 2x/4x chimera 8x

  15. Results • 225 seedlings tested (FCM) - age 5-6 months • 73 putative tetraploid plants or 32%

  16. Alternative ploidy identification methods Polyad measurements Stomatal measurements 4x stomates 12% larger • 4x polyads 20% larger Can only be used on mature flowering trees 2x 4x 2x (av length 20.9µm) 4x (av length 24.3µm)

  17. Is there a high throughput, accurate ploidy identification method with no sample preparation??????

  18. NIRANear Infrared Analysis • NIR analysis works when light usually from a halogen bulb is reflected onto the sample and the energy released from the vibration of specific bonds is converted to a spectra. • Spectral data are combined with multivariate analysis techniques such as principal components analysis to enable quantitative or qualitative analysis. • Requires expertise

  19. Instrumentation Bruker MPA Fourier Transform NIR analyser Dispersive eg PolychromixPhazir™ dispersion instrument scans the sample one wavelength at a time resolution lower no sample preparation required portable • FT instrument scans all wavelengths coincidentally, • higher resolution • no sample preparation required

  20. NIRA calibration flow chart Diverse Population set is collected Spectra are measured A calibration set selected A validation set selected FCM analysis for all samples Calibration model created using Multivariate Statistics Add “outlier” samples to Calibration set Validation testing of the model Routine Analysis Are check samples outliers? Are check samples accurate? Check wet chemistry

  21. Ploidy models Bruker MPA Fourier Transform NIR analyser Dispersive Polychromix Phazir™

  22. Ploidy score plots Bruker MPA Fourier Transform NIR analyser Dispersive Polychromix Phazir™

  23. Polyploid identification methods • 1. Flow Cytometry • expensive, slow sample preparation, assesses all layers, definitive result • 2.Stomata measurements • accuracy and cost depends on access to computer software for measurements, low throughput • only assesses epidermis • 3.Polyad measurements • only available on mature flowering trees • 4. Near Infra Red Analysis • expensive, calibration requires expertise • fresh material, can efficiently do more samples/plant • high throughput • is model robust? • 5. Chromosome counts • requires actively growing roots, harvest must not damage plant, chromosomes very small, low throughput

  24. Conclusion • Flow cytometry although labour intensive provides definitive result for ploidy analysis • becomes expensive however when more than one phyllode per plant must be tested and when follow-up testing is required to confirm stability of ploidy inductions

  25. Acknowledgements In Australia In Vietnam Dr Thinh (Director RCFTI)- lab and field staff Ms Duong Thanh Hoa, Mr Do Huu Son, Mr Le Son, Ms Nghiem Chi, Mr Tran Duc Vuong, Mr Tran Huu Bien, Mr Mai Trung Kien • School of Plant Science – UTAS • Anthony Koutoulis, Rod Griffin, Aina Price, Alvin Lam, Susan Foster, Clare Brooker • Mark Cozens – Menzies Research Institute , UTAS • Thomas Rodman – Central Science Lab, UTAS • Chris Harwood – Ecosystem Sciences, CSIRO

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